Zhong Zeng

Liver allotransplantation constitutes a vital organ-replacement therapy for end-stage liver disease, yet the global shortage of donor livers severely limits treatment capacity. Porcine liver xenotransplantation, which employs genetically edited pig livers as grafts, holds promise for alleviating donor scarcity and providing bridging organ support for patients with acute liver failure. In recent years, considerable progress has been achieved in preclinical non-human primate (NHP) models and human clinical studies. Benefiting from multi-gene editing, optimized immunosuppressive regimens, and refined surgical techniques, this approach can now effectively avert hyperacute rejection (HAR) and achieve short-term graft functional compensation. Nevertheless, clinical translation of liver xenotransplantation still faces multiple obstacles. Technical bottlenecks include delayed immune injury mediated by non-α-1,3-galactose (α-Gal) antigens, intractable coagulopathy, cross-species incompatibility in metabolism and drug disposition, lack of standardized surgical protocols, and risks of cross-species transmission of porcine viruses. Non-technical challenges encompass inconsistent global regulatory standards, informed-consent issues, animal welfare, and medical equity. Risks posed by exogenous viruses such as porcine cytomegalovirus (PCMV) can be mitigated by establishing designated pathogen-free (DPF) donor pig breeding systems combined with pre-transplant pathogen screening; however, such strategies cannot eliminate porcine endogenous retroviruses (PERV) integrated within the porcine genome, which carry risks of latent reactivation, recombination-mediated mutation, and potential cross-species infection. This review summarizes research progress and core limiting factors in liver xenotransplantation, aiming to provide theoretical references for optimizing donor gene-editing targets, improving immune-coagulation disorder prevention and control, standardizing surgical procedures, and constructing ethical supervision frameworks, so as to facilitate safe and orderly clinical translation of liver xenotransplantation.

Background: Acute allograft rejection driven by disordered CD4 ⁺ T cell alloimmunity severely limits long-term graft survival following orthotopic liver transplantation (OLT). Conventional broad-spectrum immunosuppressants cause severe systemic side effects, while therapeutic approaches relying on metabolites to induce transplant tolerance remain insufficiently explored. Isoallolithocholic acid (isoallo-LCA), a secondary bile acid produced by gut microbiota, maintains Treg homeostasis, yet its biological function and molecular mechanisms in regulating hepatic alloimmune rejection have not been clarified. Objective: To systematically explore the therapeutic efficacy of isoallo-LCA against acute liver allograft rejection and uncover its underlying molecular regulatory mechanism. Methods: An MHC-mismatched murine orthotopic liver transplantation acute rejection model was established. Recipient survival, liver pathological injury, BANFF rejection scores and serum hepatic injury indicators (ALT, AST, TBIL) were detected to evaluate the protective effect of isoallo-LCA. Flow cytometry was applied to analyze intrahepatic Th1 and Treg proportions. Transcriptomic sequencing combined with in vivo and in vitro western blot assays was used to screen and verify key signaling molecules, and an NR4A1 inhibitor was administered to conduct rescue validation experiments. Results: Isoallo-LCA intervention significantly prolonged recipient survival, alleviated intrahepatic inflammatory damage, lowered BANFF rejection grades and decreased serum ALT, AST and TBIL levels. Isoallo-LCA inhibited the differentiation of pro-inflammatory T-bet ⁺ Th1 cells and promoted the expansion of FOXP3 ⁺ Tregs, restoring the balance of intrahepatic CD4 ⁺ T cell subsets. NR4A1 was identified as the core downstream effector of isoallo-LCA. Blockade of NR4A1 pharmacologically nearly eliminated the protective phenotypes mediated by isoallo-LCA, confirming the essential role of the NR4A1/FOXP3 signaling pathway. Conclusion: Isoallo-LCA relieves acute liver allograft rejection by activating the NR4A1/FOXP3 signaling axis to remodel intrahepatic Th1/Treg immune balance. These findings provide preclinical evidence supporting gut microbial bile acid metabolites as potential low-toxic adjuvant candidates for inducing transplant immune tolerance, although clinical translation requires further validation in human studies.

The shortage of donor livers motivates the development of pig‐to‐non‐human‐primate liver xenotransplantation. While gene‐editing‐mediated xenoantigen knockout reliably prevents hyperacute rejection (HAR), nevertheless, the integrated in‐vivo phenotype and coagulation‐associated complications of multi‐gene‐edited porcine livers remain poorly characterized. To address this knowledge gap, we established orthotopic auxiliary partial liver xenotransplantation in non‐human primates using 6‐, 8‐, and 10‐gene‐edited porcine donors, and performed multi‐modal longitudinal phenotyping covering graft function assessment, immune‐coagulation profiling, PERV/PCMV DNA screening, as well as mechanistic analyses of graft injury. Consistent with prior reports, all three transplantations succeeded; xenoantigen knockout prevented HAR, and grafts maintained basal function with only mild sinusoidal congestion and limited complement‐antibody deposition. Strikingly, every recipient developed severe thrombocytopenia, graft thrombosis, and thrombotic microangiopathy, which ultimately culminated in graft dysfunction. Further analyses revealed that aberrant endothelial ASGR1 and vWF expression together with porcine macrophage‐mediated platelet clearance constituted principal drivers of platelet depletion; meanwhile, PERV and PCMV DNA were undetectable in recipient tissues. Collectively, although xenoantigen knockout effectively averts HAR, irreversible coagulation‐cascade activation, thrombosis and consumptive coagulopathy still represent the principal barriers to long‐term xenograft survival even in the setting of donor‐expressed human anticoagulant transgenes hTBM, hCD39, and hEPCR. These findings provide valuable insights for future donor‐engineering and complication‐intervention strategies.

Hepatocellular carcinoma (HCC), a highly aggressive primary liver cancer, exhibits unclear heterogeneity in RNA-binding protein (RBP) expression dynamics.

SF3B4 encodes a core subunit of the U2-type spliceosome and is implicated in abnormal cell growth and tumorigenesis. However, its role in regulating gene expression and alternative splicing in hepatocellular carcinoma (HCC) remains inadequately understood. SF3B4 expression was downregulated in HCC cells, followed by high-throughput transcriptome sequencing to capture the transcriptomic changes induced by SF3B4. This approach facilitated the identification of potential targets regulated by SF3B4 at both the transcriptional and alternative splicing levels in HCC cells. Additionally, SF3B4-binding RNAs in Huh7 cells were identified through iRIP-seq. The RNA-seq data were subsequently analyzed to elucidate the molecular mechanisms by which SF3B4 affects gene expression and alternative splicing in HCC. Downregulation of SF3B4 promoted apoptosis and inhibited cell proliferation. In the RNA-seq data, the number of regulated alternative splicing events (RASE) significantly outnumbered the differentially expressed genes (DEGs), consistent with SF3B4’s role as a splicing factor that regulates a wide array of pre-mRNA splicing events. Furthermore, a total of 252 common RNA targets bound by SF3B4 were identified. Correlation analysis with RNA-seq data suggested that SF3B4 may bind to TRIM28, potentially modulating its mRNA expression levels. Additionally, SF3B4 may influence pre-mRNA alternative splicing by interacting with SETD5. SF3B4 contributes to HCC progression by directly binding mRNAs and interacting with proteins, thereby regulating gene expression and alternative splicing.

Gene‐edited (GE) pig‐to‐human xenotransplantation continues to make breakthroughs, but which kind of gene combination is suitable for organ‐specific transplantation remains unclear. In this study, we utilised CRISPR/Cas9 gene editing technology, PiggyBac transposon system, and serial somatic cell cloning technology to develop GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene‐edited cloned (GEC) donor pigs and performed pig‐to‐non‐human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The 8‐GEC pigs were obtained by recloning with a 33‐day‐old 8‐GEC fetus with O blood type, which was generated after cell transfection, screening of cell colonies, and somatic cell cloning. Molecular identification at DNA, mRNA, and protein levels confirmed successful 8‐gene editing. Three copies of transgenes were identified by droplet digital polymerase chain reaction and whole genome sequencing, which were inserted into the introns of pig RFTN1 and MYO10 genes, as well as the intergenic region between PRLR and LOC110257300 genes of these 8‐GEC pigs. The 8‐GEC pigs also exhibited the ability of germline transmission when mated with our previously generated 4‐GEC male pigs. Moreover, antigen–antibody binding assay and complement‐dependent cytotoxicity assay demonstrated that 8‐gene editing effectively reduced the immune incompatibility and kidney xenograft from 8‐GEC pigs survived for 15 and 17 days in two NHPs, respectively. Postoperatively, the recipient serum antibodies IgA, IgG and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. Serum creatinine was normal within 10 days post operation. However, the kidney xenograft developed active antibody‐mediated rejection at necropsy, characterised by the deposition of antibodies IgG and IgM, as well as complements C4d, C3c and C5b‐C9, infiltration of CD68⁺ macrophages, and micro‐thrombotic embolism of glomerular capillaries, etc. In conclusion, we successfully developed fertile 8‐GEC pigs, which effectively alleviated immune rejection and exerted life‐supporting kidney function in the recipients.

Background: The increasing prevalence of fibrosis in donor livers raises concerns about its impact on post-transplantation outcomes, though this relationship remains unclear. This study aims to assess the effect of donor liver fibrosis on patient and graft survival following liver transplantation.Methods: Data from the UNOS-STAR registry (1987–2024) were analyzed, focusing on patients who received liver transplants with biopsy-proven fibrosis. The cohort was stratified based on fibrosis grade, and outcomes were compared using Cox regression and Kaplan-Meier survival analysis. Competing risk models were applied to assess specific mortality causes, and subgroup analyses explored the sensitivity of fibrosis on transplant outcomes.Results: Of the 22,897 patients, 17,926 received non-fibrotic grafts, and 4,971 had grafts with varying fibrosis grades. Donor fibrosis was associated with donor age, steatosis, and portal infiltrate, generally affecting those in better overall condition. Significant differences were observed in patient survival (p=0.001) and graft survival (p=0.002) between the fibrosis and non-fibrosis groups. Further analysis revealed that fibrosis increased the risk of malignancy (p=0.028), cardiovascular disease (p=0.017), and respiratory failure (p=0.033), but showed lower rejection rates at six months and one year. Sensitivity analyses confirmed fibrosis as an independent risk factor, with varying effects in subgroups.Conclusion: Donor liver fibrosis significantly impacts post-transplant outcomes, notably increasing the risk of all-cause mortality and graft failure. Specific causes of death, such as malignancy and cardiovascular disease, were more prevalent in recipients of fibrotic grafts, highlighting the need for further research to refine donor selection criteria.